Untargeted Metabolomics of Human Airway Epithelium Reveals Neuroactive Signatures Linked to Pulmonary Neuroendocrine Cell Enrichment and Allergen Exposure

<b>Background:</b> Pulmonary neuroendocrine cells (PNECs) are rare airway sensory cells implicated in amplifying allergic inflammation, yet due to their scarcity, the contribution of PNECs to the metabolic programs and responses of the airway epithelium remains poorly defined. Using a newly developed PNEC-enriched human airway epithelial model (ePNEC), we investigated the influence of PNECs on neuroendocrine and immune-modulatory metabolite production in response to the common aeroallergen of the house dust mite (HDM). <b>Methods</b>: Human bronchial epithelial cells (HBECs) and ePNEC cultures were differentiated at the air-liquid interface. Global untargeted metabolomics was performed to quantify metabolite abundance at baseline and following stimulation with HDMs. Differential expression, overlap significance, metabolite class enrichment, and pathway analyses were used to define PNEC-specific metabolic programs. <b>Results</b>: Principal component analysis (PCA) demonstrated strong baseline separation between ePNECs and HBECs, with HDMs inducing additional within-cell-type shifts. ePNECs displayed broader and more pronounced metabolite changes than HBECs. Baseline differences were largely preserved following allergen exposure, with significant overlap in both up- and down-regulated metabolites. ePNECs exhibited enriched neurotransmitter-linked metabolites-including serotonin, L-noradrenaline, dopamine, and histamine-at baseline and after HDM exposure. Amino acid-centered metabolism dominated the dataset, with enhanced histidine and tryptophan pathway activity in ePNECs. Pathway analysis revealed significant enrichment of phenylalanine, tyrosine, tryptophan, glutathione, and arginine-proline metabolism in ePNECs, whereas HBECs showed no significant pathway-level enrichment after HDM exposure. <b>Conclusions</b>: Human ePNECs engage a distinct, neuroactive metabolic program that is amplified upon HDM exposure. These findings provide a metabolic framework for how PNECs shape epithelial and neuroimmune responses to inhaled allergens.